The present invention relates to small magnetic material particles, or magnetizable beads, in fluid flows and, more particularly, to detection of such particles or beads in such flows.
Among the biomolecular detection methods used to detect selected molecules in the presence of other kinds of molecules mixed therewith are binding assays which are based on use of certain binding molecules to capture through specific chemical bondings the molecules selected for detection. Such specific bondings include polynucleic acid bondings or hybridizations involving DNA and RNA, antibody to antigen bondings, and various ligand to various receptor bondings. The detection of the selected molecules may be of primary interest in its own right, but may instead be of primary interest in indicating the presence of some other analyte molecule, species or organism.
An assay to measure the presence and concentration of such is begun by applying a sample solution containing various kinds of molecules, possibly including the molecules selected for detection, to the sensor along with label molecules attached to label magnetizable beads (or magnetic material particles) also present in the sample solution or in a supplemental solution concurrently also applied. The binding molecules through specific bondings, or recognition events, capture the selected molecules or the label molecules attached to label beads, or both, and thereafter hold them at the corresponding capture sites, i.e. the sites of the binding molecules undergoing such a bonding.
Label molecules on label beads are needed so that the occurrence of a recognition event leads to some measurable signal to indicate that a selected molecule was found present. One kind of label bead for doing this is a paramagnetic material bead having magnetizations that depend on externally applied magnetic fields. Application of such an externally applied field forcefully draws away any unbound label beads leaving the bound label beads at the capture sites while also magnetizing those bound label beads. Magnetic field detectors at the capture sites must sense the anomalies introduced into the externally applied field by the presence of bound label beads to produce the desired signals indicating the number of, and possibly the location of, such bound label beads. From this information the number of selected molecules, and kinds thereof, in the sample solution can be determined.
Such label beads can range in magnetic material composition from pure ferromagnetic material (e.g. permalloy) to small percentages of paramagnetic material encapsulated in plastic or ceramic spheres. As indicated above, such label beads are typically coated with a chemical or biological species that selectively binds to the selected molecules in an analyte of interest including DNA, RNA, viruses, bacteria, microorganisms, proteins, etc. to define the assay function, or the kind of recognition events, to be associated with that bead.
However, the label beads must typically be very small, that is, on the order of a few to tens of nanometers (nm) up to maybe a hundred or so or even up to a few microns in some instances. As a result the anomalies in an externally applied field will be very small. The capability for detection of such very small magnetic material particles in a microfluidic flow system has many uses. The presence and the motion of a particle in a flowing liquid relates to the velocity of the flow stream, to the transport of a particular species that may be connected to the particle, and to the results of any biochemical binding events occurring in connection with such flows, including with the species in such particle flows.
Detecting individual magnetic material particles in microfluidic flow streams is difficult in many such situations. Several magnetoresistive devices have been previously fabricated for this purpose. There has been some success with these devices, particularly in detecting relatively large (13×18×85 μm) segments of ferrofluids in such flows. These segments filled an entire microfluidic channel in the detection device, and were directed so as to pass over some “giant magnetoresistive” effect (GMR) magnetic fields detectors formed from two ferromagnetic layers, such as permalloy material layers, being separated by a sufficiently thin conductive layer such as copper.
A significant difficulty in extending such detection technology to the detection of individual ones of the magnetic material particles in such flows is that a single particle is not necessarily confined to a specific location in the cross section of a microfluidic channel during such flow of the ferrofluids. Thus, a 1.0 μm diameter particle may stray up to 17 μm from the bottom of an 18 μm deep channel, or it may hug the bottom, the left side, or the right side of that channel. This lack of certainty in position in the flow channel leads to drastically different magnetic field disruptions or anomalies (magnetic intensity vs. time) due to those otherwise substantially identical particles passing by the magnetic field detector. These differences in field disruption characteristics result in a smaller probability of the magnetic fields detectors being able to distinguish disruption signals due to magnetic material particles passing the detector from background magnetic fluctuation noise, and so both in making quantitative calculations of particle magnetic properties and in counting each particle separately in flows of multiple magnetic material particles. Thus, there is desired a magnetic particle detector arrangement that provides for better detection of passing magnetic particles in a liquid flow entraining to an extent such particles.